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Human cord blood-derived AC133+ progenitor cells preserve endothelial progenitor characteristics after long term in vitro expansion.

Janic B, Guo AM, Iskander AS, Varma NR, Scicli AG, Arbab AS - PLoS ONE (2010)

Bottom Line: However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients.During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers.These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, Michigan, United States of America. bjanic@rad.hfh.edu

ABSTRACT

Background: Stem cells/progenitors are central to the development of cell therapy approaches for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization. Endothelial progenitor cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). Studies showed that intravenously administered progenitors isolated from bone marrow, peripheral or cord blood home to ischemic sites. However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients. Hence, the ability to amplify the numbers of autologous EPCs by long term in vitro expansion while preserving their angiogenic potential is critically important for developing EPC based therapies. Therefore, the objective of this study was to evaluate the capacity of cord blood (CB)-derived AC133+ cells to differentiate, in vitro, towards functional, mature endothelial cells (ECs) after long term in vitro expansion.

Methodology: We systematically characterized the properties of CB AC133+ cells over the 30 days of in vitro expansion. During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers. Flow cytometry and immunocytochemistry demonstrated that CB AC133+ cells' expression of endothelial progenitor markers was not affected by long term in vitro culturing. After culturing under EC differentiation conditions, cells exhibited high expression of mature ECs markers, such as CD31, VEGFR-2 and von Willebrand factor, as well as the morphological changes indicative of differentiation towards mature ECs. In addition, throughout the 30 day culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated-acetylated-low density lipoprotein (DiI-Ac-LDL), in vitro and in vivo migration towards chemotactic stimuli and in vitro tube formation.

Conclusions: These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

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Functional analysis of long term expanded CB AC133+ cells to form tubes in Matrigel.Tube like structures after 24 h of CB AC133+ cells and HDMVECs co-culture (A-D). Complete tubes in matrigel formed by HDMVECs incubated in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h (E). When plated alone, CB AC133+ did not form tube like structures (F). Note in panels A-D, HDMVECs labeled with Calcein (C, green fluorescence) and CB AC133+ cells labeled with DiI (D, red fluorescence) co-localized (yellow; panel A) to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered between the tubes. Overlays of bright light microscopy and fluorescent microscopy images (A, C, D). Bright light microscopy only, images shown in panels B, E and F. Magnification 10x.
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pone-0009173-g006: Functional analysis of long term expanded CB AC133+ cells to form tubes in Matrigel.Tube like structures after 24 h of CB AC133+ cells and HDMVECs co-culture (A-D). Complete tubes in matrigel formed by HDMVECs incubated in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h (E). When plated alone, CB AC133+ did not form tube like structures (F). Note in panels A-D, HDMVECs labeled with Calcein (C, green fluorescence) and CB AC133+ cells labeled with DiI (D, red fluorescence) co-localized (yellow; panel A) to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered between the tubes. Overlays of bright light microscopy and fluorescent microscopy images (A, C, D). Bright light microscopy only, images shown in panels B, E and F. Magnification 10x.

Mentions: The ability of long term expanded CB AC133+ cells to respond to the extracellular matrix components by forming tubular structures was analyzed at day 10–15 and 25–30 of primary culture, by plating the cells on matrigel coated dishes for 24 h. After 24 h of co-culture with HDMVEC, CB AC133+ cells exhibited migratory and organizational pattern indicative of incorporation into the tube like structures. As shown in Figure 6A-D, HDMVECs labeled with Calcein (green fluorescence) and CB AC133+ cells labeled with DiI (red fluorescence) co-localized to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered in between the tubes. It is plausible that structural basis for the observed tube structure came from HDMVECs, with CB AC133+ playing a supporting role. To address that question, we incubated HDMVECs in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h after which HDMVECs formed complete tubes in matrigel (Figure 6E). This data indicated also the possible indirect, paracrine angiogenic role for CB AC133+ progenitors. When plated alone, CB AC133+ did not form tube like structures (Figure 6F).


Human cord blood-derived AC133+ progenitor cells preserve endothelial progenitor characteristics after long term in vitro expansion.

Janic B, Guo AM, Iskander AS, Varma NR, Scicli AG, Arbab AS - PLoS ONE (2010)

Functional analysis of long term expanded CB AC133+ cells to form tubes in Matrigel.Tube like structures after 24 h of CB AC133+ cells and HDMVECs co-culture (A-D). Complete tubes in matrigel formed by HDMVECs incubated in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h (E). When plated alone, CB AC133+ did not form tube like structures (F). Note in panels A-D, HDMVECs labeled with Calcein (C, green fluorescence) and CB AC133+ cells labeled with DiI (D, red fluorescence) co-localized (yellow; panel A) to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered between the tubes. Overlays of bright light microscopy and fluorescent microscopy images (A, C, D). Bright light microscopy only, images shown in panels B, E and F. Magnification 10x.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2820083&req=5

pone-0009173-g006: Functional analysis of long term expanded CB AC133+ cells to form tubes in Matrigel.Tube like structures after 24 h of CB AC133+ cells and HDMVECs co-culture (A-D). Complete tubes in matrigel formed by HDMVECs incubated in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h (E). When plated alone, CB AC133+ did not form tube like structures (F). Note in panels A-D, HDMVECs labeled with Calcein (C, green fluorescence) and CB AC133+ cells labeled with DiI (D, red fluorescence) co-localized (yellow; panel A) to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered between the tubes. Overlays of bright light microscopy and fluorescent microscopy images (A, C, D). Bright light microscopy only, images shown in panels B, E and F. Magnification 10x.
Mentions: The ability of long term expanded CB AC133+ cells to respond to the extracellular matrix components by forming tubular structures was analyzed at day 10–15 and 25–30 of primary culture, by plating the cells on matrigel coated dishes for 24 h. After 24 h of co-culture with HDMVEC, CB AC133+ cells exhibited migratory and organizational pattern indicative of incorporation into the tube like structures. As shown in Figure 6A-D, HDMVECs labeled with Calcein (green fluorescence) and CB AC133+ cells labeled with DiI (red fluorescence) co-localized to form tube like structure. Most of the green fluorescent cells appeared to be structural part of the tubes, while some of the red florescence cells that did not became part of the tube network remained scattered in between the tubes. It is plausible that structural basis for the observed tube structure came from HDMVECs, with CB AC133+ playing a supporting role. To address that question, we incubated HDMVECs in the presence of CB AC133+ cells' supernatants (w/o EPCs and VEGF) for 24 h after which HDMVECs formed complete tubes in matrigel (Figure 6E). This data indicated also the possible indirect, paracrine angiogenic role for CB AC133+ progenitors. When plated alone, CB AC133+ did not form tube like structures (Figure 6F).

Bottom Line: However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients.During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers.These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

View Article: PubMed Central - PubMed

Affiliation: Cellular and Molecular Imaging Laboratory, Department of Radiology, Henry Ford Hospital, Detroit, Michigan, United States of America. bjanic@rad.hfh.edu

ABSTRACT

Background: Stem cells/progenitors are central to the development of cell therapy approaches for vascular ischemic diseases. The crucial step in rescuing tissues from ischemia is improvement of vascularization that can be achieved by promoting neovascularization. Endothelial progenitor cells (EPCs) are the best candidates for developing such an approach due to their ability to self-renew, circulate and differentiate into mature endothelial cells (ECs). Studies showed that intravenously administered progenitors isolated from bone marrow, peripheral or cord blood home to ischemic sites. However, the successful clinical application of such transplantation therapy is limited by low quantities of EPCs that can be generated from patients. Hence, the ability to amplify the numbers of autologous EPCs by long term in vitro expansion while preserving their angiogenic potential is critically important for developing EPC based therapies. Therefore, the objective of this study was to evaluate the capacity of cord blood (CB)-derived AC133+ cells to differentiate, in vitro, towards functional, mature endothelial cells (ECs) after long term in vitro expansion.

Methodology: We systematically characterized the properties of CB AC133+ cells over the 30 days of in vitro expansion. During 30 days of culturing, CB AC133+ cells exhibited significant growth potential that was manifested as 148-fold increase in cell numbers. Flow cytometry and immunocytochemistry demonstrated that CB AC133+ cells' expression of endothelial progenitor markers was not affected by long term in vitro culturing. After culturing under EC differentiation conditions, cells exhibited high expression of mature ECs markers, such as CD31, VEGFR-2 and von Willebrand factor, as well as the morphological changes indicative of differentiation towards mature ECs. In addition, throughout the 30 day culture cells preserved their functional capacity that was demonstrated by high uptake of DiI fluorescently conjugated-acetylated-low density lipoprotein (DiI-Ac-LDL), in vitro and in vivo migration towards chemotactic stimuli and in vitro tube formation.

Conclusions: These studies demonstrate that primary CB AC133+ culture contained mainly EPCs and that long term in vitro conditions facilitated the maintenance of these cells in the state of commitment towards endothelial lineage.

Show MeSH
Related in: MedlinePlus